Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to scavenge such species. Oxidative stress can be caused by many different pathways, intrinsic and extrinsic, mediated either by mitochondrial respiration or by membrane-bound NADPH oxidases. ROS play an important role in the progression of several diseases including, but not limited to, inflammation, atherosclerosis, aging and age-related degenerative disorders. Probes that can detect ROS in serum samples, live tissue explants, cell cultures, and in vivo have potential uses for medical diagnostics and research tools for the diagnoses of diseases characterized by increased ROS production.
Imaging enables multiplex analysis, localization and quantitation of different parameters related to cytotoxicity and cell death in the same cell. Thus, detection of ROS by conventional fluorescence microscopy, fluorescence spectroscopy, flow cytometry, and/or high content imaging is likely to be advantageous over other techniques. Fluorescent sensors for superoxide and the hydroxyl radical, such as dihydroethidium (DHE), have been used as ROS probes. However, DHE has limited applicability due to its spontaneous auto-oxidation, rapid photobleaching, high toxicity, and multiple reaction products with ROS. Furthermore, the lower emission wavelength of DHE makes its use in vivo problematic. Dihydrorhodamine (DHR), another reduced dye that has been investigated for detection of ROS, suffers from high rates of oxidation, thereby limiting its applications. Reduced cyanine dyes developed thus far as probes for ROS, which are based on Cy3/Cy5/Cy7, suffer to varying degrees from solubility problems and/or from auto-oxidation. Sulfonate ester-based dyes have also been investigated as ROS probes. These probes, which typically require multistep synthesis procedures that are time-consuming and expensive, undergo rapid hydrolysis thereby limiting their application.
Thus, there exists a need for probes to detect ROS, amenable for use in vitro or in vivo, that do not suffer from the limitations of prior art ROS probes, such as their tendency to undergo spontaneous auto-oxidation catalyzed by oxygen and/or light with concomitant production of high levels of background fluorescence.